While wild-type GFP revolutionized the observation and monitoring of gene expression, there were several problems associated with it. It's intensity after excitation is low in many cell types, it takes time to fluoresce after protein formation, and it is expressed poorly in mulitple mammalian cell types. In order to counter these problems, various types of enhanced GFP's have been created. In one such example, 2 point mutations were made in the GFP chromophore: Ser65 to was changed to Thr and Phe64 was changed to Leu. EGFP was shown to possess many favorable characteristics over not only GFP, but also other forms of expression. Firstly, it requires no cofactors, enzymes, or additional gene products to fluoresce, moving it past non-GFP gene markers immediately. In comparison to GFP, it fluoresces 35 times brighter when excited by blue light, is more soluble, and has more efficient protein folding characteristics than wild-type GFP. In addition, the altered enzymes in EGFP are more prevalent and preferred in eukaryotic cells.<cite>Zhang1996</cite>

While wild-type GFP revolutionized the observation and monitoring of gene expression, there were several problems associated with it. It's intensity after excitation is low in many cell types, it takes time to fluoresce after protein formation, and it is expressed poorly in mulitple mammalian cell types. In order to counter these problems, various types of enhanced GFP's have been created. In one such example, 2 point mutations were made in the GFP chromophore: Ser65 to was changed to Thr and Phe64 was changed to Leu. EGFP was shown to possess many favorable characteristics over not only GFP, but also other forms of expression. Firstly, it requires no cofactors, enzymes, or additional gene products to fluoresce, moving it past non-GFP gene markers immediately. In comparison to GFP, it fluoresces 35 times brighter when excited by blue light, is more soluble, and has more efficient protein folding characteristics than wild-type GFP. In addition, the altered enzymes in EGFP are more prevalent and preferred in eukaryotic cells.<cite>Zhang1996</cite>

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[[Image:destabilizedEGFP|thumb|200px|Comparison of standard and destabilized EGFP in the presence of CHX.]]

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[[Image:destabilizedEGFP.jpg|thumb|200px|Comparison of standard and destabilized EGFP in the presence of CHX.]]

=== Destabilized GFP ===

=== Destabilized GFP ===

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GFP is extremely useful due to its stability; however, this same stability limits the applications of GFP in studies that require rapid turnover of the reporter. In order to compensate for this, various forms of destabilized GFP have been created. Amino acids 422-461 of the degradation domain of mouse ornithine decarboxylace (MODC) were added to the C-terminus of EGFP that degrades in the presence of cycloheximide (CHX). This sequence, known as the PEST sequence, has been linked with protein degradation due to correlation in C-terminal sequences of proteins with shot half-lives. The image on the right shows that in the presence of CHX, the modified EGFP (EGFP-MODC) degraded rapidly in comparison to standard EGFP.<cite>Li1998</cite>

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GFP is extremely useful due to its stability; however, this same stability limits the applications of GFP in studies that require rapid turnover of the reporter. In order to compensate for this, various forms of destabilized GFP have been created. Amino acids 422-461 of the degradation domain of mouse ornithine decarboxylace (MODC) were added to the C-terminus of EGFP that degrades in the presence of cycloheximide (CHX). This sequence, known as the PEST sequence, has been linked with protein degradation due to correlation in C-terminal sequences of proteins with shot half-lives. Modification of the PEST sequence added onto EGFP led to various rates of degradation of the protein. The image on the right shows that in the presence of CHX, the modified EGFP (EGFP-MODC) degraded rapidly in comparison to standard EGFP.<cite>Li1998</cite>

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== Luciferase ==

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Luciferases are catalysts of enzymatic reactions that cause emission of light in the visible spectrum (bioluminescence).

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=== Bacterial Luciferases ===

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Bacterial luciferases exist mostly in marine Gammaproteobacteria and have various purposes and requirements. One of the cooler functions of bioluminescent bacteria is in symbiotic relationships with higher organisms, such as that of the bacterium in the specialized organism of the angler fish.

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The most well known bacterial systems are the ''lux'' systems of the ''Photobacterium phosphoreum, P. leiognathi, Vibrio harveyi, V. fischeri, and P. luminescencs.'' All of these systems are basically encoded by the same ''luxCDABE'' system, with variations (image on the right).

Green Fluorescent Protein (GFP)

History

GFP was first discovered by Osamu Shimomura in Aequorea jellyfish as a companion protein to the aequorin responsible for the blue glow of the organism.[1][2] Shimomura and his group further characterized and identified the peak luminescence of GFP as similar to that of Aequorea tissue, both of which differed from the peak of the aequorin protein significantly, indicating that GFP altered the color of the aequorin from its natural blue to the green expressed by the organism. They showed that the mechanism for this was transfer of energy from the aequorin to GFP in the presence of a cation[3] The crucial breakthrough

came when Douglas Prasher et al cloned the gene and identified its amino acid and DNA sequence.[4] Further characterization showed that expression of the gene led to luminescence in other organism, providing the key inference that all of the information necessary for post-translational synthesis of the chromophore was in the gene itself, and no jellyfish-specific enzymes were needed for production of functional GFP.

GFP molecules drawn in cartoon style, one fully and one with the side of the beta barrel cut away to reveal the chromophore

Structure and Characterization

GFP consists of a single β-sheet with alpha helices containing the covalently bonded chromophore 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI) running through the center. The fluorescence of GFPs is dependent on the key sequence Ser-Tyr-Gly (amino acids 65–67).[5] Wild type GFP has been shown to have 2 excitation peaks at 395-397nm and 470-475nm. The emission spectrum of wild type GFP has a single peak at 504nm.

GFP Derivatives (excluding EGFP and destabilized GFPS)

As of 1998, there were 7 main classes of GFP:

wild-type mixture of neutral phenol and anionic phenolate (above)

phenolate anion

neutral phenol

phenolate anion with stacked pi electron system

indole

imidazole

phenyl

The excitation and emission wavelength spectra and the color of emission of each of these derivatives are different, as shown in the table.
The first 4 classes are polypeptides with a Tyr and position 66, while the final 3 have Trp, His and Phe at that position respectively.[2]

Enhanced GFP (EGFP)

While wild-type GFP revolutionized the observation and monitoring of gene expression, there were several problems associated with it. It's intensity after excitation is low in many cell types, it takes time to fluoresce after protein formation, and it is expressed poorly in mulitple mammalian cell types. In order to counter these problems, various types of enhanced GFP's have been created. In one such example, 2 point mutations were made in the GFP chromophore: Ser65 to was changed to Thr and Phe64 was changed to Leu. EGFP was shown to possess many favorable characteristics over not only GFP, but also other forms of expression. Firstly, it requires no cofactors, enzymes, or additional gene products to fluoresce, moving it past non-GFP gene markers immediately. In comparison to GFP, it fluoresces 35 times brighter when excited by blue light, is more soluble, and has more efficient protein folding characteristics than wild-type GFP. In addition, the altered enzymes in EGFP are more prevalent and preferred in eukaryotic cells.[6]

Comparison of standard and destabilized EGFP in the presence of CHX.

Destabilized GFP

GFP is extremely useful due to its stability; however, this same stability limits the applications of GFP in studies that require rapid turnover of the reporter. In order to compensate for this, various forms of destabilized GFP have been created. Amino acids 422-461 of the degradation domain of mouse ornithine decarboxylace (MODC) were added to the C-terminus of EGFP that degrades in the presence of cycloheximide (CHX). This sequence, known as the PEST sequence, has been linked with protein degradation due to correlation in C-terminal sequences of proteins with shot half-lives. Modification of the PEST sequence added onto EGFP led to various rates of degradation of the protein. The image on the right shows that in the presence of CHX, the modified EGFP (EGFP-MODC) degraded rapidly in comparison to standard EGFP.[5]

Luciferase

Luciferases are catalysts of enzymatic reactions that cause emission of light in the visible spectrum (bioluminescence).

Bacterial Luciferases

Bacterial luciferases exist mostly in marine Gammaproteobacteria and have various purposes and requirements. One of the cooler functions of bioluminescent bacteria is in symbiotic relationships with higher organisms, such as that of the bacterium in the specialized organism of the angler fish.

The most well known bacterial systems are the lux systems of the Photobacterium phosphoreum, P. leiognathi, Vibrio harveyi, V. fischeri, and P. luminescencs. All of these systems are basically encoded by the same luxCDABE system, with variations (image on the right).